Compositions and methods for detection of ehrlichia canis and ehrlichia chaffeensis antibodies

ABSTRACT

The invention provides methods and compositions for the detection of  Ehrlichia canis  and  Ehrlichia chaffeensis  antibodies and antibody fragments.

TECHNICAL AREA OF THE INVENTION

[0001] The invention provides compositions and methods for the detectionand quantification of Ehrlichia canis and Ehrlichia chaffeensisantibodies and antibody fragments.

BACKGROUND OF THE INVENTION

[0002] The Ehrilichia are obligate intracellular pathogens that infectcirculating lymphocytes in mammalian hosts. Ehrlichia canis andEhrlichia chaffeensis are members of the same sub-genus group thatinfect canines and humans and cause canine monocytic ehrlichiosis (CME)and human monocytic ehrlichiosis (HME), respectively. The canine diseaseis characterized by fever, lymphadenopathy, weight loss, andpancytopenia. In humans the disease is characterized by fever, headache,mylagia, and leukopenia. Early detection and treatment are important fortreating both canine and human ehrlichiosis.

[0003] Indirect immunofluorescense assays (IFA) and enzyme-linkedimmunosorbent assays (ELISA) are frequently used as aids in thediagnosis of these diseases. These assays measure or otherwise detectthe binding of anti-Ehrlichia antibodies from a patient's blood, plasma,or serum to infected cells, cell lysates, or purified Ehrlichiaproteins. However, currently known assays for detecting anti-Ehrlichiaantibodies or fragments thereof are severely limited in usefulnessbecause of sensitivity and specificity issues directly related to theimpure nature of the Ehrlichia antigen used in these tests. Highlypurified reagents are needed to construct more accurate assays.

SUMMARY OF THE INVENTION

[0004] It is an object of the invention to provide reagents and methodsfor detecting anti-Ehrlichia canis antibodies and anti-Ehrlichiachaffeensis antibodies. This and other objects of the invention areprovided by one or more of the embodiments described below.

[0005] One embodiment of the invention provides a composition of mattercomprising an isolated polypeptide selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, and variants thereof.

[0006] Another embodiment of the invention provides a composition ofmatter comprising an isolated polypeptide selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof, and a carrier.

[0007] Still another embodiment of the invention provides a method ofdetecting the presence of antibodies to Ehrlichia. The method comprisescontacting one or more polypeptides selected from the group consistingof the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof,with a test sample suspected of comprising antibodies to Ehrlichia,under conditions that allow polypeptide/antibody complexes to form. Thepolypeptide/antibody complexes are detected. The detection ofpolypeptide/antibody complexes is an indication that antibodies toEhrlichia are present in the test sample.

[0008] Yet another embodiment of the invention provides a devicecontaining one or more polypeptides selected from the group consistingof the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof,and instructions for use of the one or more polypeptides for theidentification of an Ehrlichia infection in a mammal.

[0009] Still another embodiment of the invention provides an article ofmanufacture comprising packaging material and, contained within thepackaging material, one or more polypeptides selected from the groupconsisting of the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and variantsthereof. The packaging material comprises a label that indicates thatthe one or more polypeptides can be used for the identification ofEhrlichia infection in a mammal.

[0010] Even another embodiment of the invention provides a method ofdiagnosing an Ehrlichia infection in a mammal. The method comprisesobtaining a biological sample from a mammal suspected of having anEhrlichia infection, and contacting one or more polypeptides selectedfrom the group consisting of the polypeptides shown in SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, and variants thereof, with the biological sample under conditionsthat allow polypeptide/antibody complexes to form. Polypeptide/antibodycomplexes are detected, wherein the detection of polypeptide/antibodycomplexes is an indication that the mammal has an Ehrlichia infection.

[0011] Another embodiment of the invention provides a monoclonalantibody that specifically binds to at least one epitope of an Ehrlichiacanis or Ehrlichia chaffeensis polypeptide selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0012] The invention therefore provides highly purified polypeptides andantibodies for use in accurate assays for the detection of Ehrlichiaantibodies and antibody fragments.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Polypeptides of the Invention

[0014] The invention provides highly purified reagents for the detectionof E. canis and E. chaffeensis antibodies and antibody fragments. Inparticular, the invention provides polypeptides having at least 85%identity, more preferably at least 90% identity, and still morepreferably at least 96%, 97%, 98%, or 99% identity to a polypeptidesequence shown in SEQ ID NOs:1-7. See Table 1. Polypeptides that do notcomprise 100% identity to a polypeptide sequence shown in SEQ ID NOs:1-7are considered “variants,” and are considered polypeptides of theinvention.

[0015] The E. canis peptides were identified using phage displaytechnology by determining the amino acid sequence bound by a mousemonoclonal antibody (IIIH7) raised against native E. canis antigen. TheIIIH7 monoclonal antibody was used to affinity purify virus-expressingpeptides in a PDH 10 phage display library. The sequences or mimetopesbound by IIIH7 demonstrated strong sequence homology to outer membraneproteins of E. canis and E. chaffeensis. The outer membrane proteins ofboth species are encoded by a polymorphic gene family, which results inmultiple reproductions of the proteins. TABLE 1 Peptide Derived SEQ IDNO Sequence of Peptide From SEQ ID NO: 1 KSTVGVFGLKHDWDGSPILK E. canisP30-1 SEQ ID NO: 2 NTTTGVFGLKQDWDGATIKD E. canis P30 SEQ ID NO: 3NTTVGVFGLKQNWDGSAISN E. chaffeensis P28 SEQ ID NO: 4 NPTVALYGLKQDWNGVSAE. chaffeensis OMP-1C SEQ ID NO: 5 NTTVGVFGIEQDWDRCVIS E. chaffeensisOMP-1D SEQ ID NO: 6 NPTVALYGLKQDWEGISS E. chaffeensis OMP-1E SEQ ID NO:7 NTTTGVFGLKQDWDGSTIS E. chaffeensis OMP-1F

[0016] Identity means amino acid sequence similarity and has an artrecognized meaning. Sequences with identity share identical or similaramino acids, where similar amino acids are preferably conserved aminoacids. Conserved amino acids are amino acids that possess similar sidechains and properties (e.g., hydrophilic, hydrophobic, aromatic) as theamino acids encoded by the reference sequence. Thus, a candidatesequence sharing 85% amino acid sequence identity with a referencesequence (i.e., SEQ ID NOs:1-7) requires that, following alignment ofthe candidate sequence with the reference sequence, 85% of the aminoacids in the candidate sequence are identical to the corresponding aminoacids in the reference sequence, and/or constitute conservative aminoacid changes.

[0017] Sequences are aligned for identity calculations using amathematical algorithm, such as the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlinand Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:403-410. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences with identity to the polypeptides of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST) can beused. Internal gaps and amino acid insertions in the candidate sequenceas aligned are ignored when making the identity calculation.

[0018] Variants in which amino acids of the polypeptides of theinvention are substituted, deleted, or added in any combination arecontemplated by the invention. Naturally occurring variants andnon-naturally occurring variants are included in the invention and maybe produced by mutagenesis techniques or by direct synthesis.

[0019] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Suchvariants include deletions, insertions, inversions, repeats, andsubstitutions selected according to general rules known in the art so ashave little effect on activity. For example, guidance concerning how tomake phenotypically silent amino acid substitutions is provided in Bowieet al., Science, 247:1306-1310 (1990), wherein the authors indicate thatthere are two main strategies for studying the tolerance of an aminoacid sequence to change.

[0020] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, the amino acidpositions which have been conserved between species can be identified.These conserved amino acids are likely important for protein function.In contrast, the amino acid positions in which substitutions have beentolerated by natural selection indicate positions which are not criticalfor protein function. Thus, positions tolerating amino acid substitutionmay be modified while still maintaining specific binding activity of thepolypeptide to anti-Ehrlichia antibodies or antibody fragments.

[0021] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site-directed mutagenesis oralanine-scanning mutagenesis (the introduction of single alaninemutations at every residue in the molecule) can be used (Cunningham etal., Science, 244:1081-1085 (1989)). The resulting mutant molecules canthen be tested for specific binding to anti-Ehrlichia antibodies orantibody fragments.

[0022] According to Bowie et al., these two strategies have revealedthat proteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,the most buried or interior (within the tertiary structure of theprotein) amino acid residues require nonpolar side chains, whereas fewfeatures of surface or exterior side chains are generally conserved.Moreover, tolerated conservative amino acid substitutions involvereplacement of the aliphatic or hydrophobic amino acids Ala, Val, Leuand Ile; replacement of the hydroxyl residues Ser and Thr; replacementof the acidic residues Asp and Glu; replacement of the amide residuesAsn and Gln; replacement of the basic residues Lys, Arg, and His;replacement of the aromatic residues Phe, Tyr, and Trp; and replacementof the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

[0023] Besides conservative amino acid substitution, variants of thepresent invention include: (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code; (ii)substitution with one or more of amino acid residues having asubstituent group; (iii) fusion of the mature polypeptide with anothercompound, such as a compound to increase the stability and/or solubilityof the polypeptide (e.g., polyethylene glycol); (iv) fusion of thepolypeptide with additional amino acids, such as an IgG Fc fusion regionpeptide, a leader or secretory sequence, or a sequence facilitatingpurification. Such variant polypeptides are deemed to be within thescope of those skilled in the art from the teachings herein.

[0024] Polypeptides of the invention specifically bind to ananti-Ehrlichia antibody. In this context “specifically binds” means thatthe polypeptide recognizes and binds to an anti-Ehrlichia antibody, butdoes not substantially recognize and bind other molecules in a testsample.

[0025] Polypeptides of the invention comprise at least one epitope thatis recognized by an anti-Ehrlichia antibody. An epitope is an antigenicdeterminant of a polypeptide. An epitope can be a linear, sequentialepitope or a conformational epitope. Epitopes within a polypeptide ofthe invention can be identified by several methods. See, e.g., U.S. Pat.No. 4,554,101; Jameson & Wolf, CABIOS 4:181-186 (1988). For example, apolypeptide of the invention can be isolated and screened. A series ofshort peptides, which together span the entire polypeptide sequence, canbe prepared by proteolytic cleavage. By starting with, for example,20-mer polypeptide fragments, each fragment can be tested for thepresence of epitopes recognized in, for example, an enzyme-linkedimmunosorbent assay (ELISA). In an ELISA assay a polypeptide, such as a20-mer polypeptide fragment, is attached to a solid support, such as thewells of a plastic multi-well plate. A population of antibodies arelabeled, added to the solid support and allowed to bind to the unlabeledantigen, under conditions where non-specific adsorbtion is blocked, andany unbound antibody and other proteins are washed away. Antibodybinding is detected by, for example, a reaction that converts acolorless indicator reagent into a colored reaction product.Progressively smaller and overlapping fragments can then be tested froman identified 20-mer to map the epitope of interest.

[0026] Preferably, a polypeptide of the invention is synthesized usingconventional peptide sythesizers, which are well known in the art. Apolypeptide of the invention can also be produced recombinantly. Apolynucleotide encoding an Ehrlichia polypeptide can be introduced intoan expression vector that can be expressed in a suitable expressionsystem using techniques well known in the art. A variety of bacterial,yeast, plant, mammalian, and insect expression systems are available inthe art and any such expression system can be used. Optionally, apolynucleotide encoding an Ehrlichia polypeptide can be translated in acell-free translation system.

[0027] If desired, an Ehrlichia polypeptide can be produced as a fusionprotein, which can also contain other amino acid sequences, such asamino acid linkers or signal sequences, as well as ligands useful inprotein purification, such as glutathione-S-transferase, histidine tag,and staphylococcal protein A. More than one Ehrlichia polypeptide can bepresent in a fusion protein. If desired, various combinations ofEhrlichia polypeptides from different ehrilichia strains or isolates canbe included in a fusion protein.

[0028] A polypeptide of the invention can be synthesized such that itcomprises several repeated Ehrlichia polypeptides. This is a multimericpolypeptide. These repeated polypeptides can comprise one specificpolypeptide, e.g. the polypeptide shown in SEQ ID NO:1, repeated 2 ormore times. Alternatively, the repeated polypeptides can comprise one ormore copies of a specific Ehrlichia polypeptide along with one or morecopies of another different Ehrlichia polypeptide. A polypeptide of theinvention can be combined or synthesized with one or more polypeptides,fragments of polypeptides, or full-length polypeptides. Preferably theone or more polypeptides are other polypeptides of the invention orother Ehrlichia proteins.

[0029] Polypeptides of the invention can also comprise fragments of thepolypeptides shown in SEQ ID NO:1-7, or variants thereof. For example,fragments of polypeptides can comprise any number of amino acids between6 and 20 amino acids.

[0030] A polypeptide of the invention is preferably combined with acarrier. A carrier is a vehicle for a polypeptide of the invention.Carriers include, for example, excipients, diluents, adjuvants, andstabilizers. Examples of such stabilizers are proteins such as serumalbumins and gelatin; saccharides such as glucose, sucrose, lactose,maltose, trehalose, sorbitol, maltitol, mannitol and lactitol; andbuffers which are mainly composed of phosphate or succinate.

[0031] Various strains and isolates of Ehrlichia canis and Ehrlichiachaffeensis occur, and polypeptides of any of these strains and isolatescan be used in the present invention. Nucleic acid and amino acidsequences of Ehrlichia genes and polypeptides are known in the art. Forexample, several sequences of the E. chaffeensis OMP gene family andseveral sequences of the E. canis P30 gene family are disclosed in WO99/13720.

[0032] Methods of Detection

[0033] The methods of the invention detect Ehrlichia canis or Ehrlichiachaffeensis antibodies or antibody fragments in a test sample, such as abiological sample, an environmental sample, or a laboratory sample. Abiological sample can include, for example, sera, blood, cells, plasma,or tissue from a mammal such as a dog or a human. The test sample can beuntreated, precipitated, fractionated, separated, diluted, concentrated,or purified before combining with a polypeptide of the invention.

[0034] The methods comprise contacting a polypeptide of the inventionwith a test sample under conditions that allow a polypeptide/antibodycomplex to form. The formation of a complex between the polypeptide andanti-Ehrlichia antibodies in the sample is detected. In one embodimentof the invention, the polypeptide/antibody complex is detected when anindicator reagent, such as an enzyme, which is bound to the antibody,catalyzes a detectable reaction. Optionally, an indicator reagentcomprising a signal generating compound can be applied to thepolypeptide/antibody complex under conditions that allow formation of apolypeptide/antibody/indicator complex. Thepolypeptide/antibody/indicator complex is detected. Optionally, thepolypeptide or antibody can be labeled with an indicator reagent priorto the formation of a polypeptide/antibody complex. The method canoptionally comprise a positive or negative control.

[0035] Assays of the invention include, but are not limited to thosebased on competition, direct reaction or sandwich-type assays. Assayscan use solid phases or substrates or can be performed byimmunoprecipitation or any other methods which do not utilize solidphases. Where a solid phase or substrate is used, a polypeptide of theinvention is directly or indirectly attached to a solid support or asubstrate such as a microtiter well, magnetic bead, non-magnetic bead,column, matrix, membrane, fibrous mat composed of synthetic or naturalfibers (e.g., glass or cellulose-based materials or thermoplasticpolymers, such as, polyethylene, polypropylene, or polyester), sinteredstructure composed of particulate materials (e.g., glass or variousthermoplastic polymers), or cast membrane film composed ofnitrocellulose, nylon, polysulfone or the like (generally synthetic innature). A preferred substrate is sintered, fine particles ofpolyethylene, commonly known as porous polyethylene, for example, 10-15micron porous polyethylene from Chromex Corporation. All of thesesubstrate materials may be used in suitable shapes, such as films,sheets, or plates, or they may be coated onto or bonded or laminated toappropriate inert carriers, such as paper, glass, plastic films, orfabrics. Suitable methods for immobilizing peptides on solid phasesinclude ionic, hydrophobic, covalent interactions and the like.

[0036] Polypeptides of the invention can be used to detectanti-Ehrlichia antibodies or antibody fragments in assays including, butnot limited to enzyme linked immunosorbent assay (ELISA), western blot,IFA, radioimmunoassay (RIA), hemagglutination (HA), and fluorescencepolarization immunoassay (FPIA). A preferred assay of the invention isthe reversible flow chromatographic binding assay, for example a SNAP®assay. See U.S. Pat. No. 5,726,010.

[0037] In one type of assay format, one or more polypeptides can becoated on a solid phase or substrate. A test sample suspected ofcontaining anti-Ehrlichia antibodies is incubated with an indicatorreagent comprising a signal generating compound conjugated to anantibody specific for Ehrlichia for a time and under conditionssufficient to form antigen/antibody complexes of either antibodies ofthe test sample to the polypeptides of the solid phase or the indicatorreagent compound conjugated to an antibody specific for Ehrlichia to thepolypeptides of the solid phase. The reduction in binding of theindicator reagent conjugated to an anti-Ehrlichia antibody to the solidphase can be quantitatively measured. A measurable reduction in thesignal compared to the signal generated from a confirmed negativeEhrlichia test sample indicates the presence of anti-Ehrlichia antibodyin the test sample. This type of assay can quantitate the amount ofanti-Ehrlichia antibodies in a test sample.

[0038] In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. A polypeptide of theinvention is conjugated to an indicator reagent and added to a testsample. This mixture is applied to the support or substrate. IfEhrlichia antibodies are present in the test sample they will bind thepolypeptide conjugated to an indicator reagent and to the polypeptideimmobilized on the support. The polypeptide/antibody/indicator complexcan then be detected. This type of assay can quantitate the amount ofanti-Ehrlichia antibodies in a test sample.

[0039] The formation of a polypeptide/antibody complex or apolypeptide/antibody/indicator complex can be detected by radiometric,colormetric, fluorometric, size-separation, or precipitation methods.Optionally, detection of the polypeptide/antibody complex is by theaddition of a secondary antibody that is coupled to a indicator reagentcomprising a signal generating compound. Indicator reagents comprisingsignal generating compounds (labels) associated with apolypeptide/antibody complex can be detected and include chromogenicagents, catalysts such as enzymes, fluorescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol,radioactive elements, direct visual labels, as well as cofactors,inhibitors, magnetic particles, and the like. Examples of enzymesinclude alkaline phosphatase, horseradish peroxidase,beta-galactosidase, and the like. The selection of a particular label isnot critical, but it will be capable of producing a signal either byitself or in conjunction with one or more additional substances.

[0040] Formation of the complex is indicative of the presence of anti-E.canis or anti-E. chaffeensis antibodies in a test sample. Therefore, themethods of the invention can be used to diagnose E. canis or E.chaffeensis infection in a patient. Each polypeptide of the inventioncan detect E. canis or E. chaffeensis or both due to cross-reactivity ofthe polypeptides and antibodies.

[0041] The methods of the invention can also indicate the amount orquantity of anti-Ehrlichia antibodies in a test sample. With manyindicator reagents, such as enzymes, the amount of antibody present isproportional to the signal generated. Depending upon the type of testsample, it can be diluted with a suitable buffer reagent, concentrated,or contacted with a solid phase without any manipulation. For example,it usually is preferred to test serum or plasma samples which previouslyhave been diluted, or concentrate specimens such as urine, in order todetermine the presence and/or amount of antibody present.

[0042] The invention further comprises assay kits for detectinganti-Ehrlichia antibodies in a sample. A kit comprises one or morepolypeptides of the invention and means for determining binding of thepolypeptide to Ehrlichia antibodies in the sample. A kit can comprise adevice containing one or more polypeptides of the invention andinstructions for use of the one or more polypeptides for theidentification of an Ehrlichia infection in a mammal. The kit can alsocomprise packaging material comprising a label that indicates that theone or more polypeptides of the kit can be used for the identificationof Ehrlichia infection. Other components such as buffers, controls, andthe like, known to those of ordinary skill in art, may be included insuch test kits. The polypeptides, assays, and kits of the invention areuseful, for example, in the diagnosis of individual cases of Ehrlichiainfection in a patient, as well as epidemiological studies of Ehrlichiaoutbreaks.

[0043] Polypeptides and assays of the invention can be combined withother polypeptides or assays to detect the presence of Ehrlichia alongwith other organisms. For example, polypeptides and assays of theinvention can be combined with reagents that detect heartworm and/orBorrelia burgdorferi.

[0044] Monoclonal Antibodies

[0045] The polypeptides of the invention can also be used to developmonoclonal and/or polyclonal antibodies that specifically bind to animmunological epitope of E. canis or E. chaffeensis present in thepolypeptides of the invention.

[0046] The antibodies or fragments thereof can be employed in assaysystems, such as a reversible flow chromatographic binding assay, enzymelinked immunosorbent assay, western blot assay, or indirectimmunofluorescense assay, to determine the presence, if any, ofEhrlichia polypeptides in a test sample. In addition, these antibodies,in particular monoclonal antibodies, can be bound to matrices similar toCNBr-activated Sepharose and used for the affinity purification ofspecific Ehrlichia proteins from, for example, cell cultures or bloodserum, such as to purify recombinant and native Ehrlichia antigens andproteins. The monoclonal antibodies of the invention can also be usedfor the generation of chimeric antibodies for therapeutic use, or othersimilar applications.

[0047] Monoclonal antibodies directed against Ehrlichia epitopes can beproduced by one skilled in the art. The general methodology forproducing such antibodies is well-known and has been described in, forexample, Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press Inc., Boca Raton, Fla. (1982), as well as thattaught by L. T. Mimms et al., Virology 176:604-619 (1990). Immortalantibody-producing cell lines can be created by cell fusion, and also byother techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus.

[0048] The following are provided for exemplification purposes only andare not intended to limit the scope of the invention described in broadterms above. All references cited in this disclosure are incorporatedherein by reference.

EXAMPLES Example 1

[0049] Detection of E. canis Antibodies in Canine Serum

[0050] The performance of a synthetic peptide SNAP® assay was comparedto the performance of a commercially available E. canis SNAP® assay thatuses partially purified E. canis antigens. The partially purified nativeantigens were obtained from E. canis organisms grown in tissue cultureand partially purified by differential centrifugation and columnchromatography. The synthetic peptides used in the synthetic peptideSNAP® assay were monomeric forms of the E. canis P30-1 or the E. canisP-30 peptide, SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0051] A population of 70 suspected E. canis positive canine samples wasobtained from Arizona, Texas, and Arkansas and tested using thesynthetic peptide SNAP® assay and the native antigen SNAP® assay. Thesamples were also tested using an indirect IFA. Briefly, the IFA assaywas performed using E. canis infected cells coated onto IFA slides andfluorescein isothiocyanate (FITC)-labeled rabbit anti-canine IgG. E.canis was harvested from cell cultures, diluted in buffer and coatedonto IFA slides. Dilutions of test samples were made in buffer,incubated with the coated IFA slides, and then washed and incubated withFITC-labeled anti-canine conjugate. Slides were washed and viewed bywith ultraviolet light microscopy. IFA results are recorded as a titerof fluorescence activity. This represents the last sample dilutionreactive on the IFA slide. Samples with IFA titers greater than or equalto 1:100 are positive.

[0052] In the case of discrepant results, an E. canis western blot wasused as the confirmatory assay. Briefly, E. canis antigen was harvestedfrom tissue culture, resolved by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and then transferred to a nitrocellulose membrane. Aftertransfer, the membrane was blocked with heterologous protein overnightat 4 degrees C. Diluted test samples of canine E. canis Ab-positive andnegative serum samples were incubated with blots for 2 hours at roomtemperature. Blots were then washed, incubated with commercialanti-canine IgG:peroxidase conjugate reagents for 1 hour and washed.Signals were developed by incubation of strips with a commercialperoxidase indicator reagent. Reaction to the immunodominat band with amolecular weight of 30,000 Daltons was required for positive resultconfirmation by western blot. See, e.g., Suksawat et al. J. Vet.Internal Med. 14:50-55 (2000).

[0053] The synthetic peptide SNAP® assay and native antigen SNAP® assaycomprised an assay system similar to that described in U.S. Pat. No.5,726,010. Briefly, a test sample is applied to a reverse flowchromatographic binding assay device and allowed to flow along andsaturate a flow matrix. This facilitates sequential complex formation.That is, an Ehrlichia antibody in the test sample binds first to annon-immobilized labeled specific binding reagent. In the case of thesynthetic peptide SNAP® assay the non-immobilized labeled specificbinding reagent is a polypeptide of the invention conjugated tohorseradish peroxidase. For the native antigen SNAP® assay the reagentcomprises partially purified native antigens. This complex binds to animmobilized analyte capture reagent. For the synthetic peptide SNAP®assay the immobilized analyte capture reagent is one or morepolypeptides of the invention conjugated to bovine serum albumin. Forthe native antigen SNAP® assay the capture reagent is partially purifiednative antigens. An absorbent reservoir is contacted with the saturatedflow matrix, thereby reversing the fluid flow. Detector and washsolution is delivered to the flow matrix. The liquid reagents removeunbound sample and unbound labeled specific binding reagent andfacilitate detection of analyte complexes at the location of the of theimmobilized analyte capture reagent. The substrate used in theseexperiments was 3,3′,5,5′tetramethylbenzidine (TMB).

[0054] Results

[0055] The results of the assays are shown in Table 2. The results canbe broken into five groups.

[0056] Group 1 comprises forty-seven samples that were positiveaccording to the synthetic peptide SNAP® assay, the native antigen SNAP®assay, and the IFA. These are antibody positive samples and noadditional testing was done on these samples.

[0057] Group 2 comprises ten samples (numbers 15, 17, 18, 20, 22, 23,24, 41, 42, and 46) that were positive according to the syntheticpeptide SNAP® assay, negative according to the native antigen SNAP®assay, positive on the IFA, and confirmed by western blot analysis.These are true positive samples that were positive on the syntheticpeptide SNAP® assay and false negative on the native antigen SNAP®assay.

[0058] Group 3 comprises five samples (numbers 1, 2, 3, 4, and 5) thatwere positive according to IFA and confirmed negative by western blotanalysis. These are true negative samples that were false positive inthe IFA. All 5 of these samples were correctly identified as negative bythe synthetic peptide SNAP® assay. The native antigen SNAP® assaycorrectly identified three of the samples (numbers 1, 2, and 5) asnegative, but gave false positive results for two samples (numbers 3 and4).

[0059] Group 4 comprises seven samples (6, 7, 8, 9, 10, 11, and 12) thatwere negative by IFA and confirmed positive by western blot analysis.These are positive samples that were false negatives in the IFA. Allseven samples were true positive on the synthetic peptide SNAP® assay.The native antigen SNAP® assay correctly identified only two of theseven samples (numbers 7 and 11) as positive and incorrectly identifiedfive of the samples (numbers 6, 8, 9, 10, and 12) resulting in falsenegative results for these five samples.

[0060] Group 5 comprises one sample (number 21) that was positive by IFAand confirmed as positive by western blot analysis. The syntheticpeptide SNAP® assay and the native antigen SNAP® assay gave negativeresults. This is a positive sample that was false negative on both thesynthetic peptide SNAP® assay and the native antigen SNAP® assay.

[0061] Therefore, 70 samples were tested and 65 of these samples weretrue positive samples. The synthetic peptide SNAP® assay correctlyidentified 64 of the positive samples for a sensitivity of 98.5%(64/65). The native antigen SNAP® assay correctly identified 49 of thesamples for a sensitivity of 75.3% (49/65). Of the five true negativesamples, the synthetic peptide SNAP® assay correctly identified 5 of thenegative samples for a specificity of 100% (5/5). The native antigenSNAPS assay correctly identified 3 of the negative samples for aspecificity of 60% (3/5). Therefore, the synthetic peptide SNAP® assayis more sensitive and specific than the native antigen SNAP® assay.TABLE 2 E. canis Ab Positive Canine Population Comparison of NativeAntigen SNAP Assay with Synthetic Peptide SNAP Assay Native Synthetic AgAssay Peptide Assay E. canis 291JS 358HT & 359HT IFA Western No. SampleI.D. E. canis H. Worm E. canis H. Worm Titer ≧ Blot 1 F119894-6 − − − −1:100 − 2 F103638-5 − − − − 1:100 − 3 2815:89E + − − − 1:100 − 4 31365 +− − − 1:100 − 5 F107158-1 − − − − 1:400 − 6 31285 − − + − − + 7 31508 +− + − − + 8 31364 − − + − − + 9 31037 − − + − − + 10 31492 − − + − − +11 28963 + − + − − + 12 31398 − − + − − + 13 31527 + − + − 1:100 + 1431552 + − + − 1:100 + 15 31556 − − + − 1:100 + 16 F101938-4 + − + −1:100 + 17 28404 − − + − 1:100 + 18 F102890-0 − − + − 1:100 + 19 31496 +− + − 1:100 not done 20 29825 − − + − 1:400 + 21 F099609-2 − − − −1:400 + 22 F121120-6 − − + − 1:400 + 23 F104088-9 − − + − 1:400 + 24F120923-5 − − + − 1:400 + 25 31368 + − + − 1:400 not done 26 31159 + − +− 1:400 not done 27 2815:89A + − + − 1:500 not done 28 2815:89B + + + +1:500 not done 29 2815:89C + − + − 1:500 not done 30 2815:89D + − + −1:500 not done 31 30597 + − + − 1:1600 not done 32 30448 + − + − 1:1600not done 33 29938 + − + − 1:1600 not done 34 31500 + − + − 1:1600 notdone 35 31249 + − + − 1:1600 not done 36 31369 + − + − 1:1600 not done37 31523 + − + − 1:1600 not done 38 31021 + − + − 1:1600 not done 3930846 + − + − 1:1600 not done 40 31536 + − + − 1:1600 not done 41F102996-1 − − + − 1:1600 + 42 F118620-1 − − + − 1:1600 + 43 F104581-1 +− + − 1:1600 not done 44 P127 + + + + 1:1600 not done 45 29363 + − + −1:1600 not done 46 F120001-5 − − + − 1:3200 + 47 F107100-7 + − + −1:3200 not done 48 F119153-3 + − + − 1:3200 not done 49 F120513-8 + − +− 1:3200 not done 50 F118601-4 + − + − 1:3200 not done 51 F121073-7 +− + − 1:3200 not done 52 2898:62 + − + − 1:3200 not done 53 28392 + − +− 1:3200 not done 54 29375 + − + − 1:3200 not done 55 29099 + − + −1:3200 not done 56 28580 + − + − 1:3200 not done 57 28960 + − + − 1:3200not done 58 29361 + − + − 1:3200 not done 59 30864 + − + − 1:6400 notdone 60 31158 + − + − 1:6400 not done 61 31169 + − + − 1:6400 not done62 28094 + − + − 1:6400 not done 63 28098 + − + − 1:6400 not done 6428174 + − + − 1:6400 not done 65 28513 + − + − 1:6400 not done 6628830 + − + − 1:6400 not done 67 28846 + − + − 1:6400 not done 6828914 + − + − 1:6400 not done 69 17101 + − + − 1:6400 not done 7021120 + − + − 1:6400 not done

We claim:
 1. A composition of matter comprising an isolated polypeptideselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and variantsthereof.
 2. The composition of claim 1, further comprising a carrier. 3.A method of detecting presence of antibodies to Ehrlichia comprising:(a) contacting one or more polypeptides selected from the groupconsisting of the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and variantsthereof, with a test sample suspected of comprising antibodies toEhrlichia, under conditions that allow polypeptide/antibody complexes toform; (b) detecting polypeptide/antibody complexes; wherein thedetection of polypeptide/antibody complexes is an indication thatantibodies to Ehrlichia are present in the test sample.
 4. The method ofclaim 3, further comprising contacting the complexes of step (a) with anindicator reagent comprising a signal generating compound that generatesa measurable signal prior to the performance of step (b).
 5. The methodof claim 3, wherein the presence of antibodies to Ehrlichia canis aredetected.
 6. The method of claim 3, wherein the presence of antibodiesto Ehrlichia chaffeensis are detected.
 7. The method of claim 3, whereinthe antibodies are fragments of antibodies.
 8. The method of claim 3wherein the amount of antibody in a test sample is determined.
 9. Themethod of claim 3, wherein the polypeptide is attached to a substrate.10. The method of claim 3, wherein the polypeptide provided is shown inSEQ ID NO:1.
 11. The method of claim 3, wherein the polypeptide providedis shown in SEQ ID NO:2.
 12. The method of claim 3, wherein thepolypeptide provided is shown in SEQ ID NO:3.
 13. The method of claim 3,wherein the polypeptide provided is shown in SEQ ID NO:4.
 14. The methodof claim 3, wherein the polypeptide provided is shown in SEQ ID NO:5.15. The method of claim 3, wherein the polypeptide provided is shown inSEQ ID NO:6.
 16. The method of claim 3, wherein the polypeptide providedis shown in SEQ ID NO:7.
 17. The method of claim 3, wherein the one ormore polypeptides are provided in a multimeric form.
 18. The method ofclaim 3, wherein the test sample is a biological sample obtained from amammal.
 19. The method of claim 18, wherein the mammal is selected fromthe group consisting of humans and dogs.
 20. The method of claim 3wherein the method comprises an assay selected from the group of assaysconsisting of a reversible flow chromatographic binding assay, an enzymelinked immunosorbent assay, a western blot assay, and an indirectimmunofluorescense assay.
 21. A device containing one or morepolypeptides selected from the group consisting of the polypeptidesshown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof.
 22. The device ofclaim 21, further comprising instructions for use of the one or morepolypeptides for the identification of an Ehrlichia infection in amammal.
 23. The device of claim 22, wherein the identification of anEhrlichia infection is done using a method of detecting presence ofantibodies to Ehrlichia comprising: (a) contacting one or morepolypeptides selected from the group consisting of the polypeptidesshown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof, with a test samplesuspected of comprising antibodies to Ehrlichia, under conditions thatallow polypeptide/antibody complexes to form; (b) detectingpolypeptide/antibody complexes; wherein the detection ofpolypeptide/antibody complexes is an indication that an Ehrlichiainfection is present.
 24. The device of claim 22, wherein the Ehrlichiainfection is caused by Ehrlichia canis or Ehrlichia chaffeensis.
 25. Anarticle of manufacture comprising packaging material and, containedwithin the packaging material, one or more polypeptides selected fromthe group consisting of the polypeptides shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,and variants thereof.
 26. The article of manufacture of claim 25 whereinthe packaging material comprises a label that indicates that the one ormore polypeptides can be used for the identification of Ehrlichiainfection in a mammal.
 27. The article of manufacture of claim 26,wherein the identification of an Ehrlichia infection is done using amethod of detecting presence of antibodies to Ehrlichia comprising: (a)contacting one or more polypeptides selected from the group consistingof the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof,with a test sample suspected of comprising antibodies to Ehrlichia,under conditions that allow polypeptide/antibody complexes to form; (b)detecting polypeptide/antibody complexes; wherein the detection ofpolypeptide/antibody complexes is an indication that an Ehrlichiainfection is present.
 28. The article of manufacture of claim 26,wherein the Ehrlichia infection is caused by Ehrlichia canis orEhrlichia chaffeensis.
 29. A method of diagnosing an Ehrlichia infectionin a mammal comprising: (a) obtaining a biological sample from a mammalsuspected of having an Ehrlichia infection; (b) contacting one or morepolypeptides selected from the group consisting of the polypeptidesshown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and variants thereof, with thebiological sample under conditions that allow polypeptide/antibodycomplexes to form; (c) detecting polypeptide/antibody complexes; whereinthe detection of polypeptide/antibody complexes is an indication thatthe mammal has an Ehrlichia infection.
 30. The method of claim 29further comprising contacting the complexes of step (b) with anindicator reagent comprising a signal generating compound that generatesa measurable signal prior to the performance of step (c).
 31. The methodof claim 29, wherein the Ehrlichia infection is caused by Ehrlichiacanis.
 32. The method of claim 29, wherein the Ehrlichia infection iscaused by Ehrlichia chaffeensis.
 33. The method of claim 29, wherein themammal is a human or a dog.
 34. A monoclonal antibody that specificallybinds to at least one epitope of an Ehrlichia canis or Ehrlichiachaffeensis polypeptide, said polypeptide selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7.